Mutation-screening High Yield L-lactic Acid Strain

L-lactic acid is an important organic acid, widely used in food, medicine, chemical industry. Particularly, because of the developing of biodegradable polymers, such as, ploy-lactic acid, much L-Lactic acid was demanded.In this paper, the strain of Lactobacillus casei CICC6028 was chosen as the original strain. The media and culture technology were optimized and the results showed that when the seed culture time was 24h, inoculum size was 10%, pH was 6.8, culture temperature was 34℃and the fermentation medium composition was as (g/L): Glucose 140,Beef Extract 9,Yeast Extract 4.5,Peptone 9,Anhydrous Sodium Acetate 5,MgSO₄·7H₂O 0.2,MnSO₄ 0.125, Twain-80 1mL, CaCO₃ 50, the yield of L-Lactic acid was reached to 90.7g/L.By studying the ultraviolet, microwave, diethyl sulfate, low energy nitrogen ion beam and other mutagen mutagenic effects on the strains showed that the optimal mutation parameters were: UV lamp power 15W, the sample distance from the tube 15cm, treatment time 15s; microwave power 480W, intermittent treatment method,and total time 40s; diethyl sulfate content of 1%, treatment time 30min; nitrogen ion beam energy 10Kev, dose 50×2.6×10¹³. The best of the mutation parameters were used to treat the strain Lactobacillus casei CICC6028. By repeated mutation and screening, the highest yield L-lactic acid mutant strain which named NE-19 was obtained, the lactic acid yield of the mutant strain reached to 121.3g/L, increased by 33.7% compared with the original strain. By studying the different temperatures effects on the yield of L-lactic acid of the mutant strain, it was found, when the culture temperature was 40℃, the yield of L-Lactic acid was highest. Indicating that the best fermentation temperature of the mutant strain was 40℃. High Performance Liquid Metabolite was used to analyze the production of the mutant strain it was found that, the production of the mutant was lactic acid and there were no other by-products almost.The fermentation medium of the mutant was optimized by Response Surface Method and the optimum medium was as flowing (g/L): Glucose 153.7, peptone 17.3, Beef Extract 15.9, Yeast Extract 8.3, Anhydrous Sodium Acetate 5, MgSO₄·7H₂O 0.2, MnSO4 0.125, Twain-80 1mL, CaCO₃ 50, pH 6.8。The averaged lactic acid yield of the mutant strain was 134.00±2.59g/L when the optimized medium was used.By comparing the fermentation curves of mutant strain and the original strain, it was found that, the L-lactic acid accumulation and sugar consumption of the mutant strain was faster than that of original strain. When the fermentation time reached to 120h, the lactic acid production of the mutant strain got to 142g/L, and the residual sugar was 3.6g/L, while, lactic acid production of the original strain was only 98g/L and the residual sugar content was as high as 43.7g/L. It suggested that, the mutant was an ideal strain for L-Lactic acid producing.